129Xe NMR biosensors represent a fundamentally new class of biophysical probes with tremendous potential as cancer diagnostic agents. The proposed studies build on a Xe biosensor program that has been continuously funded (PI: Dmochowski) for the past 10 years by DoD, NIH R21, R33, and R01 grants. NIH R01 renewal funding is now requested to continue this dynamic and highly productive program. A focus of this research program is the development of 129Xe MRI contrast agents for improved diagnosis of lung cancer. To date, we have made key advances in the synthesis, xenon affinity, hyperpolarized (hp) 129Xe NMR spectroscopy, and biological application of Xe biosensors utilizing a cryptophane moiety for Xe encapsulation. The development of next-generation 129Xe MRI contrast agents is rapidly advancing, now propelled by recent improvements in 129Xe hyperpolarization technology. An 'open source' system produces near-unity polarization in ~1-L quantities required for human lung imaging. The Dmochowski laboratory will gain access to a state-of the-art xenon polarizer within the next two years, with support from S10 funding (PI: Rizi). This proposal focuses on a 129Xe NMR technique employing chemical exchange saturation transfer ('Hyper-CEST'), which was pioneered using cryptophane as the xenon host by the Pines lab at Berkeley in 2006, and incorporates concepts of xenon polarization transfer contrast (XTC) first described by Mugler and Ruppert at Virginia in 2000. In 2012, our laboratory showed that 1 picomolar cryptophane provides useful contrast using Hyper-CEST NMR, a 109-fold sensitivity enhancement over standard MRI contrast agents. This improved upon the original 5 nM cryptophane detection sensitivity reported at Berkeley, and is still roughly 100-fold more sensitive than Hyper-CEST measurements performed for single-site cryptophane entities by researchers in France and Germany. We have been able to attribute only some of these differences in Hyper-CEST efficiency to the greater Xe affinity and faster Xe exchange kinetics of our trifunctionalized, water-soluble cryptophanes. This raises several important questions: What is the operative mechanism for small molecule-mediated 129Xe magnetization transfer? Can these processes be optimized to achieve femtomolar (or better) detection sensitivity? Can small molecule and genetically encoded xenon-binding CEST agents be developed for wide distribution to labs interested in molecular imaging? To address the first question, we hypothesize that a Xe bubble surrounds the cryptophane, with many weakly-associated, exterior Xe atoms undergoing rapid magnetization transfer at short-range with the single interior Xe atom. This hypothesis will be rigorously tested by computational and experimental approaches in Aim 1.1, working with UPenn Chemistry collaborator Saven. While cryptophanes enable explorations of xenon biosensing, their scarcity limits use to a handful of labs worldwide. Thus, in Aim 1.2 we propose to develop new small-molecule Hyper-CEST agents that can be widely distributed for biomedical research. Our lab made the recent discovery that commercially available cucurbituril CB[6] can be detected at 1 picomolar concentration via Hyper-CEST NMR, similar to water-soluble cryptophane. Moreover, we determined that CB[6] can be detected by 129Xe NMR in cells and cell lysate. One shortcoming of CB[6] is the difficulty of functionalizing this host molecule with single targeting moieties. To overcome this problem, we will develop turn on CB[6] xenon biosensors that exploit the affinity of CB[6] for many organic small molecules. As with cryptophane, we will seek to elucidate and improve upon CB[6] Hyper-CEST contrast by computational and experimental approaches. Our lab will develop water-soluble cryptophane and CB[6] solutions for targeting lung cancer cells, and perform Hyper-CEST NMR spectroscopy and imaging studies. In Aim 2, we propose the development of genetically encoded MRI analogs of green fluorescent protein (GFP) and color variants, which are the current standard for visualizing many cellular processes by fluorescence microscopy. Cellular production of GFP increases the spatial and temporal information encoded by this fluorophore, and also circumvents many problems of cell delivery, localization, and degradation. Similarly, protein-based xenon biosensors will expand the repertoire of cellular and in vivo studies, while taking advantage of the much greater tissue penetration of MRI relative to light microscopy. A recent report of gas vesicle (GV) proteins that achieve Hyper-CEST provides useful precedent. GVs, however, are composed of 8-14 different proteins that self-assemble in bacteria but cannot be expressed in eukaryotic cells. Thus, we are focused on developing more versatile single-protein Hyper-CEST agents. MD simulations published by the Geissler laboratory led us to hypothesize correctly that beta-lactamase should enable Hyper-CEST contrast, based on its large number of cryptic allosteric sites that provide ~1-nanometer hydrophobic pockets in the protein interior where Xe may transiently reside. In collaboration with Temple collaborators (Carnevale, Klein), in Aim 2.1, we will study Xe interactions with beta-lactamase using several computational approaches, and develop variants of beta-lactamase that increase CEST contrast, while also enabling multiplexing experiments (similar to CFP, GFP, YFP, RFP for fluorescence microscopy). In Aim 2.2, we will perform Hyper-CEST NMR spectroscopy and imaging studies using beta-lactamase variants.